Voltage-Dependent Gating of Single Sodium Channels from Mammalian Neuroblastoma Cells
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Facilitation of Rabbit ב1B Calcium Channels: Involvement of Endogenous Gגד Subunits
Keywords: Calcium channel, G protein, Facilitation 7535 Journal of Physiology (1998), 509.1, pp.15—27 15 Facilitation of rabbit á1B calcium channels: involvement of endogenous Gâã subunits Gary J. Stephens, Nicola L. Brice, Nicholas S. Berrow and Annette C. Dolphin Department of Pharmacology, University College London and Royal Free Hospital School of Medicine, London WC1E 6BT, UK (Received 30 October 1997; accepted after revision 26 January 1998) 1. The á1B (N-type) calcium channel shows strong G protein modulation in the presence of G protein activators or Gâã subunits. Using transient expression in COS_7 cells of á1B together with the accessory subunits áµ—ä and â2a,wehaveexaminedtheroleofendogenous Gâã subunits in the tonic modulation of á1B, and compared this with modulation by exogenously expressed Gâã subunits. 2. Prepulse facilitation of control á1BÏáµ—äÏâ2a currents was always observed. This suggests the existenceoftonicmodulationofá1B subunits. To determine whether endogenous Gâã is involved in the facilitation observed in control conditions, the âARK1 Gâã-binding domain (amino acids 495—689) was overexpressed, in order to bind free Gâã subunits. The extent of control prepulse-induced facilitation was significantly reduced, both in terms of current amplitude and the rate of current activation. In agreement with this, GDPâS also reduced the control facilitation. 3. Co-expression of the GâÔãµ subunit, together with the á1BÏáµ—äÏâ2a calcium channel combination, resulted in a marked degree of depolarizing prepulse-reversible inhibition of the whole-cell ICa or IBa. Both slowing of current activation and inhibition of the maximum current amplitude were observed, accompanied by a depolarizing shift in the mid-point of the voltage dependence of activation. -
Chapter 25 Mechanisms of Action of Antiepileptic Drugs
Chapter 25 Mechanisms of action of antiepileptic drugs GRAEME J. SILLS Department of Molecular and Clinical Pharmacology, University of Liverpool _________________________________________________________________________ Introduction The serendipitous discovery of the anticonvulsant properties of phenobarbital in 1912 marked the foundation of the modern pharmacotherapy of epilepsy. The subsequent 70 years saw the introduction of phenytoin, ethosuximide, carbamazepine, sodium valproate and a range of benzodiazepines. Collectively, these compounds have come to be regarded as the ‘established’ antiepileptic drugs (AEDs). A concerted period of development of drugs for epilepsy throughout the 1980s and 1990s has resulted (to date) in 16 new agents being licensed as add-on treatment for difficult-to-control adult and/or paediatric epilepsy, with some becoming available as monotherapy for newly diagnosed patients. Together, these have become known as the ‘modern’ AEDs. Throughout this period of unprecedented drug development, there have also been considerable advances in our understanding of how antiepileptic agents exert their effects at the cellular level. AEDs are neither preventive nor curative and are employed solely as a means of controlling symptoms (i.e. suppression of seizures). Recurrent seizure activity is the manifestation of an intermittent and excessive hyperexcitability of the nervous system and, while the pharmacological minutiae of currently marketed AEDs remain to be completely unravelled, these agents essentially redress the balance between neuronal excitation and inhibition. Three major classes of mechanism are recognised: modulation of voltage-gated ion channels; enhancement of gamma-aminobutyric acid (GABA)-mediated inhibitory neurotransmission; and attenuation of glutamate-mediated excitatory neurotransmission. The principal pharmacological targets of currently available AEDs are highlighted in Table 1 and discussed further below. -
Current, TTX-Resistant Na؉ Current, and Ca2؉ Current in the Action Potentials of Nociceptive Sensory Neurons
The Journal of Neuroscience, December 1, 2002, 22(23):10277–10290 Roles of Tetrodotoxin (TTX)-Sensitive Na؉ Current, TTX-Resistant Na؉ Current, and Ca2؉ Current in the Action Potentials of Nociceptive Sensory Neurons Nathaniel T. Blair and Bruce P. Bean Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 20114 Nociceptive sensory neurons are unusual in expressing carry the largest inward charge during the upstroke of the voltage-gated inward currents carried by sodium channels re- nociceptor action potential (ϳ58%), with TTX-sensitive sodium sistant to block by tetrodotoxin (TTX) as well as currents carried channels also contributing significantly (ϳ40%), especially near by conventional TTX-sensitive sodium channels and voltage- threshold, and high voltage-activated calcium currents much dependent calcium channels. To examine how currents carried less (ϳ2%). Action potentials had a prominent shoulder during by each of these helps to shape the action potential in small- the falling phase, characteristic of nociceptive neurons. TTX- diameter dorsal root ganglion cell bodies, we voltage clamped resistant sodium channels did not inactivate completely during cells by using the action potential recorded from each cell as the action potential and carried the majority (58%) of inward the command voltage. Using intracellular solutions of physio- current flowing during the shoulder, with high voltage-activated logical ionic composition, we isolated individual components of calcium current also contributing significantly (39%). -
An Unexpected Role for Brain-Type Sodium Channels in Coupling of Cell Surface Depolarization to Contraction in the Heart
An unexpected role for brain-type sodium channels in coupling of cell surface depolarization to contraction in the heart Sebastian K. G. Maier*, Ruth E. Westenbroek*, Kenneth A. Schenkman†, Eric O. Feigl‡, Todd Scheuer*, and William A. Catterall*§ Departments of *Pharmacology, †Pediatrics, and ‡Physiology and Biophysics, University of Washington, Seattle, WA 98195 Contributed by William A. Catterall, December 27, 2001 ␣ Voltage-gated sodium channels composed of pore-forming and ventricular myocytes compared with Nav1.5, but they contribute auxiliary  subunits are responsible for the rising phase of the significantly to coupling of cell surface depolarization to con- action potential in cardiac muscle, but the functional roles of traction because of their location in the transverse tubules. Our distinct sodium channel subtypes have not been clearly defined. results provide the first evidence, to our knowledge, for a unique Immunocytochemical studies show that the principal cardiac pore- functional role of TTX-sensitive brain-type sodium channels in forming ␣ subunit isoform Nav1.5 is preferentially localized in the heart. intercalated disks, whereas the brain ␣ subunit isoforms Nav1.1, Nav1.3, and Nav1.6 are localized in the transverse tubules. Sodium Methods currents due to the highly tetrodotoxin (TTX)-sensitive brain iso- All procedures were approved by the Institutional Animal Care forms in the transverse tubules are small and are detectable only and Use Committee of the University of Washington. after activation with  scorpion toxin. Nevertheless, they play an important role in coupling depolarization of the cell surface mem- Cell Isolation. Ventricular myocytes were isolated from adult male brane to contraction, because low TTX concentrations reduce left (8–10 weeks) wild-type B6129F1 mice, as described (14). -
The Role of S4 Charges in Voltage-Dependent
ARTICLE The Role of S4 Charges in Voltage-dependent and Voltage-independent KCNQ1 Potassium Channel Complexes Gianina Panaghie and Geoffrey W. Abbott Greenberg Division of Cardiology, Department of Medicine and Department of Pharmacology, Cornell University, Weill Medical College, New York, NY 10021 Voltage-gated potassium (Kv) channels extend their functional repertoire by coassembling with MinK-related peptides (MiRPs). MinK slows the activation of channels formed with KCNQ1 α subunits to generate the voltage- dependent IKs channel in human heart; MiRP1 and MiRP2 remove the voltage dependence of KCNQ1 to generate potassium “leak” currents in gastrointestinal epithelia. Other Kv α subunits interact with MiRP1 and MiRP2 but without loss of voltage dependence; the mechanism for this disparity is unknown. Here, sequence alignments re- vealed that the voltage-sensing S4 domain of KCNQ1 bears lower net charge (+3) than that of any other eukaryotic voltage-gated ion channel. We therefore examined the role of KCNQ1 S4 charges in channel activation using alanine-scanning mutagenesis and two-electrode voltage clamp. Alanine replacement of R231, at the N-terminal side of S4, produced constitutive activation in homomeric KCNQ1 channels, a phenomenon not observed with previous single amino acid substitutions in S4 of other channels. Homomeric KCNQ4 channels were also made constitutively active by mutagenesis to mimic the S4 charge balance of R231A-KCNQ1. Loss of single S4 charges at positions R231 or R237 produced constitutively active MinK-KCNQ1 channels and increased the constitutively active component of MiRP2-KCNQ1 currents. Charge addition to the CO2H-terminal half of S4 eliminated constitu- tive activation in MiRP2-KCNQ1 channels, whereas removal of homologous charges from KCNQ4 S4 produced constitutively active MiRP2-KCNQ4 channels. -
ACTX-Hv1c, Isolated from the Venom of the Blue Mountains
S.J. Gunning et al. Janus-faced atracotoxins block KCa channels The Janus-faced atracotoxins are specific blockers of invertebrate KCa channels Simon J. Gunning1, Francesco J. Maggio2,4, Monique J. Windley1, Stella M. Valenzuela1, Glenn F. King3 and Graham M. Nicholson1 1 Neurotoxin Research Group, Department of Medical & Molecular Biosciences, University of Technology, Sydney, Broadway, NSW 2007, Australia 2 Department of Molecular, Microbial & Structural Biology, University of Connecticut School of Medicine, Farmington, Connecticut 06032, USA and 3 Division of Chemical and Structural Biology, Institute for Molecular Bioscience, University of Queensland, Brisbane QLD 4072, Australia 4 Current affiliation: Bristol-Myers Squibb, 6000 Thompson Road, Syracuse NY 13057, USA Subdivision: Molecular Neurobiology Abbreviations: ACTX, atracotoxin; 4-AP, 4-aminopyridine; BKCa channel, 2+ + 2+ large-conductance Ca -activated K channel; CaV channel, voltage-activated Ca channel; ChTx, charybdotoxin; DRG, dorsal root ganglia; DUM, dorsal unpaired median; EGTA, ethylene glycol-bis(b-aminoethyl ether)-N,N,N’N’-tetraacetic acid; IbTx, + iberiotoxin; J-ACTX, Janus-faced atracotoxin; KA channel, transient ‘A-type’ K channel; 2+ + + KCa channel, Ca -activated K channel; KDR channel, delayed-rectifier K channel; KV + + channel, voltage-activated K channel; NaV channel, voltage-activated Na channel; Slo, Slowpoke; TEA, tetraethylammonium; TTX, tetrodotoxin. RUNNING TITLE: Janus-faced atracotoxins block KCa channels ADDRESS CORRESPONDENCE TO: Graham M. Nicholson, Department of Medical & Molecular Biosciences, University of Technology, Sydney, PO Box 123, Broadway NSW 2007, Australia. Tel: +61 2 9514-2230 Fax: +61 2 9514-2228; E-mail: [email protected] 1 S.J. Gunning et al. Janus-faced atracotoxins block KCa channels ABSTRACT The Janus-faced atracotoxins are a unique family of excitatory peptide toxins that contain a rare vicinal disulfide bridge. -
Conditional Knockout of Nav1.6 in Adult Mice Ameliorates Neuropathic Pain
www.nature.com/scientificreports OPEN Conditional knockout of NaV1.6 in adult mice ameliorates neuropathic pain Received: 22 November 2017 Lubin Chen 1,2,3, Jianying Huang1,2,3, Peng Zhao1,2,3, Anna-Karin Persson1,2,3, Accepted: 19 February 2018 Fadia B. Dib-Hajj1,2,3, Xiaoyang Cheng1,2,3, Andrew Tan1,2,3, Stephen G. Waxman1,2,3 & Published: xx xx xxxx Sulayman D. Dib-Hajj 1,2,3 Voltage-gated sodium channels NaV1.7, NaV1.8 and NaV1.9 have been the focus for pain studies because their mutations are associated with human pain disorders, but the role of NaV1.6 in pain is less understood. In this study, we selectively knocked out NaV1.6 in dorsal root ganglion (DRG) neurons, using NaV1.8-Cre directed or adeno-associated virus (AAV)-Cre mediated approaches, and examined the specifc contribution of NaV1.6 to the tetrodotoxin-sensitive (TTX-S) current in these neurons and its role in neuropathic pain. We report here that NaV1.6 contributes up to 60% of the TTX-S current in large, and 34% in small DRG neurons. We also show NaV1.6 accumulates at nodes of Ranvier within the neuroma following spared nerve injury (SNI). Although NaV1.8-Cre driven NaV1.6 knockout does not alter acute, infammatory or neuropathic pain behaviors, AAV-Cre mediated NaV1.6 knockout in adult mice partially attenuates SNI-induced mechanical allodynia. Additionally, AAV-Cre mediated NaV1.6 knockout, mostly in large DRG neurons, signifcantly attenuates excitability of these neurons after SNI and reduces NaV1.6 accumulation at nodes of Ranvier at the neuroma. -
Uhm Phd 9118021 R.Pdf
INFORMATION TO USERS The most advanced technology has been used to photograph and reproduce this manuscript from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand corner and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6" x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. U·M·I University Microfilms International A Bell & Howell Information Company 300 North Zeeb Road. Ann Arbor. M148106·1346 USA 313/761-4700 800/521-0600 Order Number 9118021 Sodium channel activation mechanisms: Insights from deuterium oxide and ddta-9-tetrahydrocannabinol substitution Alicata, Daniel Andrew, Ph.D. -
Phenytoin Inhibits the Persistent Sodium Current in Neocortical Neurons by Modifying Its Inactivation Properties
Phenytoin Inhibits the Persistent Sodium Current in Neocortical Neurons by Modifying Its Inactivation Properties Elisa Colombo1, Silvana Franceschetti1, Giuliano Avanzini1, Massimo Mantegazza1,2* 1 Department of Neurophysiopathology – Epilepsy Center, Foundation IRCCS Neurological Institute C. Besta, Milan, Italy, 2 Institut de Pharmacologie Mole´culaire et Cellulaire (IPMC), CNRS UMR7275 and University of Nice-Sophia Antipolis, Valbonne, France Abstract + The persistent Na current (INaP) is important for neuronal functions and can play a role in several pathologies, although it is + small compared to the transient Na current (INaT). Notably, INaP is not a real persistent current because it undergoes inactivation with kinetics in the order of tens of seconds, but this property has often been overlooked. Na+ channel blockers, drugs used for treating epilepsy and other diseases, can inhibit INaP, but the mechanism of this action and the conditions in which INaP can be actually inhibited have not been completely clarified yet. We evaluated the action of phenytoin (PHT), a + prototype anti-epileptic Na channel blocker, on INaP inactivation in pyramidal neurons of rat sensorimotor cortical slices at different concentrations, from 5 to 100 mM. PHT did not modify INaP evoked with depolarizing voltage ramps of 50 or 21 21 100 mVs , but decreased INaP evoked by slower voltage ramps (10 mVs ). However, at all of the tested concentrations, PHT decreased INaP evoked by faster ramps when they were preceded by inactivating pre-pulses. Moreover, PHT shifted towards negative potentials the voltage-dependence of INaP inactivation and accelerated its kinetics of development also at depolarized potentials (+40 mV), not consistently with a simple inactivated state stabilizer. -
Week 5 Lecture Slides (PDF)
Why Does Cocaine Cause Fatal Heart Attacks? • Mental stimulation: Cocaine encourages vigorous activity • Sympathetic stimulation: Cocaine speeds heart rate directly • Vasoconstriction: Impairs blood flow to heart • Numbing: Blocks signals in heart 1 The Action Potential • How does it work? • Epilepsy and anticonvulsants • Local anesthetics (e.g. Novocaine) • Alcohol and alcohol antagonists • Shock therapies 2 Don’t worry • Don’t worry – this is the most confusing, complex, and technical bit of the whole class. I promise we won’t do this again. • This is important, so please pay attention and ask questions whenever you don’t understand. 3 Plan of action: • Electric cells? What? • How batteries work • The four batteries inside every neuron • Channels opening and closing • Putting it all together: The Action Potential 4 The potassium battery (The resting potential) • Draw it on the board (outside cell on top, draw leak channels) • Which way does the diffusion force point? • Which way does the electric force point? • What equilibrium does it eventually settle into? 5 Ion concentrations in and around axons: Concentration Concentration Ratio E (at 37 C, Ion ion outside (in mM) inside (in mM) Out:In in mV) K+ 5 100 1 : 20 -80 Na+ 150 15 10 : 1 +62 10,000 : Ca++ 2 0.0002 1 +123 Cl- 150 13 11.5 : 1 -65 6 What happens if we open and close channels? • Whenever you open a channel, you pull the voltage CLOSER to that channel’s REVERSAL POTENTIAL. The reversal potential is simply the Nernst potential for the one ion in question if only one ion passes through the channel. -
Saxitoxin and Tetrodotoxin. Electrostatic Effects on Sodium Channel Gating Current in Crayfish Axons
SAXITOXIN AND TETRODOTOXIN. ELECTROSTATIC EFFECTS ON SODIUM CHANNEL GATING CURRENT IN CRAYFISH AXONS STEVEN T. HEGOENESS AND JOHN G. STARKUS Department ofPhysiology, John A. Burns School ofMedicine, and the Bekesy Laboratory of Neurobiology, Pacific Biomedical Research Center, University ofHawaii, Honolulu, Hawaii 96822 ABSTRACT The effects of extracellular saxitoxin (STX) and tetrodotoxin (TTX) on gating current (I,ON) were studied in voltage clamped crayfish giant axons. At a holding potential (VH) of -90 mV, integrated gating charge (QON) was found to be 56% suppressed when 200 nM STX was added to the external solution, and 75% suppressed following the addition of 200 nM TTX. These concentrations of toxin are sufficiently high to block >99% of sodium channels. A smaller suppression of ION was observed when 1 nM STX was used (KD- 1-2 nM STX). The suppression of I,ON by external toxin was found to be hold potential dependent, with only minimal suppression observed at the most hyperpolarized hold potentials, - 140 to - 120 mV. The maximal effect of these toxins on ION was observed at hold potentials where the QON vs. VH plot was found to be steepest, -100 to -80 mV. The suppression of ION induced by TTX is partially relieved following the removal of fast inactivation by intracellular treatment with N-bromoacetamide (NBA). The effect of STX and TTX on IgON is equivalent to a hyperpolarizing shift in the steady state inactivation curve, with 200 nM STX and 200 nM TTX inducing shifts of4.9 ± 1.7 mV and 10.0 ± 2.1 mV, respectively. Our results are consistent with a model where the binding of toxin displaces a divalent cation from a negatively charged site near the external opening of the sodium channel, thereby producing a voltage offset sensed by the channel gating apparatus. -
Acid-Sensing Ion Channel 3 Matches the Acid-Gated Current in Cardiac Ischemia-Sensing Neurons
Acid-sensing ion channel 3 matches the acid-gated current in cardiac ischemia-sensing neurons Stephani P. Sutherland*, Christopher J. Benson, John P. Adelman, and Edwin W. McCleskey The Vollum Institute, Oregon Health Sciences University, Portland, OR 97201-3098 Edited by Denis Baylor, Stanford University School of Medicine, Stanford, CA, and approved October 26, 2000 (received for review August 23, 2000) Cardiac afferents are sensory neurons that mediate angina, pain ically (about 10-fold) greater amplitude in the sympathetics (7). The that occurs when the heart receives insufficient blood supply for its currents are blocked by amiloride and pass Naϩ better than Kϩ. metabolic demand (ischemia). These neurons display enormous This behavior of the currents identifies them as being carried by acid-evoked depolarizing currents, and they fire action potentials acid-sensing ion channels (ASICs) and distinguishes them from in response to extracellular acidification that accompanies myo- another type of proton-gated channel, vanilloid receptors. Krish- ϩ cardial ischemia. Here we show that acid-sensing ion channel 3 tal’s group identified ASICs as several kinetically distinct, Na - ϩ (ASIC3), but no other known acid-sensing ion channel, reproduces selective, Ca2 -permeant currents in sensory neurons that are the functional features of the channel that underlies the large evoked by lowered pH and are blocked by amiloride (13–16). acid-evoked current in cardiac afferents. ASIC3 and the native Lazdunski’s group found that the currents are carried by a proton- sensitive subfamily of channels within the larger family of epithelial channel are both especially sensitive to pH, interact similarly with ϩ Ca2؉, and gate rapidly between closed, open, and desensitized Na channels and degenerins of Caenorhabditis elegans (17).